6 replies to this topic

[poonamkd]

Dear Friends,
  I m optimizing SyBr qPCR assay for M.tb diagnosis with ABI Stepone instrument. . My primer is 123bp from IS6110 target. I should ideally get a Tm peak at 85 for positive (which i m getting in positive control DNA) samples. But everytime i m also getting a major peak at 81 in all the samples including negative, but not in positive control (H37Rv). I had tried everything right from chaniging the new set of primers, water, ordered new Dye also.... but with each new experiment i get a major peak at 81 and sometime along with that a minor peak at 84 too. I had titrated primer also and i am using 100nM, tried with 250nM which was equally good conc.
Pls suggest the reason behind getting peak at 81 how to remove that peak.
I m attaching melt curve peak.

Attached Thumbnails

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[Rsm]

Did you run a gel after RT-PCR to check for correct size of the product? It looks like you have two different products amplified. To make really sure, you can also sequence your product.
It seems that your amplification is not very efficient. Have you tried different primers? That might help as well...
Cheers,
Minna

[avi123]

In my melting curve I have one pick for all samples but they all have a  different  height's . I don't know why.

Attached Thumbnails

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[Rsm]

avi123, on Jul 13 2009, 05:43 PM, said:

In my melting curve I have one pick for all samples but they all have a  different  height's . I don't know why.

You seem to have different amounts of products: the more product, the higher the peak.
Cheers,
Minna

[avi123]

Minna, on Jul 13 2009, 07:44 PM, said:

avi123, on Jul 13 2009, 05:43 PM, said:

In my melting curve I have one pick for all samples but they all have a  different  height's . I don't know why.

You seem to have different amounts of products: the more product, the higher the peak.
Cheers,
Minna

Thanks Minna

[Ytje]

poonamkd, on Jul 11 2009, 10:13 AM, said:

Dear Friends,
  I m optimizing SyBr qPCR assay for M.tb diagnosis with ABI Stepone instrument. . My primer is 123bp from IS6110 target. I should ideally get a Tm peak at 85 for positive (which i m getting in positive control DNA) samples. But everytime i m also getting a major peak at 81 in all the samples including negative, but not in positive control (H37Rv). I had tried everything right from chaniging the new set of primers, water, ordered new Dye also.... but with each new experiment i get a major peak at 81 and sometime along with that a minor peak at 84 too. I had titrated primer also and i am using 100nM, tried with 250nM which was equally good conc.
Pls suggest the reason behind getting peak at 81 how to remove that peak.
I m attaching melt curve peak.

The highest peak (on the right) seems to be your product. The curves with high expression have no 'shoulder' on the left. The lower the expression, the higher the shoulder.
I had this once and tried designing other primers but I couldn't get rid of it. Our RT-PCR expert said it could have something to do with GC-rich streches in the amplicon (resulting in uneven melting of the product).
I desided to use the primers but always check the melting curve. If expression is high enough, there is no shoulder and then the data can be used. However, if expression is to low and the shoulder appears, I do not use the data.

Ytje

[Ytje]

Minna, on Jul 14 2009, 04:44 AM, said:

avi123, on Jul 13 2009, 05:43 PM, said:

In my melting curve I have one pick for all samples but they all have a  different  height's . I don't know why.

You seem to have different amounts of products: the more product, the higher the peak.
Cheers,
Minna

I agree with Minna.

Ytje